Process for the enantioselective enzymatic reduction of hydroxy keto compounds

ABSTRACT

In a process for the enantioselective enzymatic reduction of a hydroxy ketone of general formula I 
                         
wherein R 1 =C 1 -C 6  alkyl and R 2 =—Cl, —CN, —OH, —H or C 1 -C 6  alkyl, into a chiral diol of general formula II
 
                         
wherein R 1  and R 2  have the same meaning as in formula I, the hydroxy ketone is reduced with an oxidoreductase in the presence of NADH or NADPH as a cofactor, wherein
 
a) the hydroxy ketone is provided in the reaction at a concentration of ≧50 g/l,
 
b) the oxidized cofactor NAD or NADP having formed is regenerated continuously by oxidation of a secondary alcohol of general formula R X R Y CHOH, wherein R X , R Y  independently represent hydrogen, branched or unbranched C 1 -C 8 -alkyl and C total ≧3, and
 
c) the reduction of the hydroxy ketone and the oxidation of the secondary alcohol are catalyzed by the same oxidoreductase.

The invention relates to a process for the enantioselective enzymaticreduction of a hydroxy ketone of general formula I

wherein R₁=C₁-C₆ alkyl and R₂=—Cl, —CN, —OH, —H or C₁-C₆ alkyl, into achiral diol of general formula II

wherein R₁ and R₂ have the same meaning as in formula I, with thehydroxy ketone being reduced with an oxidoreductase in the presence of acofactor.

Chiral diols of general formula II are important intermediates in theproduction of pharmaceutical products, in particular in the productionof HMG-CoA reductase inhibitors. Such chiral diols are, for example,tert. butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate or tert. butyl(5S,3R)-3,5,6-trihydroxy-hexanoate.

Diols of this kind are produced in particular by enantioselectivereduction of the corresponding hydroxy ketones of formula I such as,e.g., tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate, tert. butyl(5S)-6-chloro-5-hydroxy-3-oxohexanoate or tert. butyl(5S)-5,6-dihydroxy-3-oxohexanoate. In doing so, the chemically catalyzedreduction has the disadvantage that, on the one hand, it may result inbyproducts due to the harsh reaction conditions and, on the other hand,yields unsatisfactory enantiomeric and diastereomeric excesses,respectively, and is technically feasible only with very large efforts.

For this reason, there have, for quite some time, been endeavours todevelop biocatalytic processes which allow for the enantioselectivereduction of the above-mentioned hydroxy ketones. Biocatalytic processesusually operate under mild conditions, which is why they can be expectedto enable the reduction of 5-hydroxy-3-oxohexanoate derivatives, whichare rather unstable anyway, without the formation of further byproducts.So far, however, it has not been possible to find any suitablebiocatalysts by means of which the enzymatic reduction of theabove-mentioned 5-hydroxy-3-oxohexanoate derivatives is feasible in aneffective manner and with isolated enzymes.

For instance, the U.S. patent specification U.S. Pat. No. 6,001,615 andthe international patent application WO 01/85975 A1 describe thereduction of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate and oftert. butyl (5S)-5,6-dihydroxy-3-oxohexanoate, respectively, withvarious yeasts of the genus Beauvaria, Pichia, Candida, Kluyveromycesand Torulaspora. Thereby, however, the conversions occur only with wholecells of wild strains and therefore can only be carried out at very lowconcentrations of far below 5%. So far, it has not yet been possible toidentify the enzymes and DNA sequences responsible for the conversions.

Furthermore, microbial conversions of structurally similar compounds aredescribed in EP 0 569 998 B 1. Therein, it has even been possible topurify an NADH-dependent enzyme from Acinetobacter calcoaceticus ATCC33305, which is also used in an isolated state together with glucosedehydrogenase for coenzyme regeneration. In the process described, thesubstrate is used at concentrations of 1% and a “total turn over number”of the NADH of merely 10 is achieved. An industrially applicable processhas not been presented.

In the U.S. patent specification U.S. Pat. No. 6,645,746 B1, an aminoacid sequence from Candida magnoliae is disclosed, which may be used forreducing tert. butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate to tert.butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate with the aid of NADPH. Inthe specification of said document, the enzyme is preferably used in astate in which it is coexpressed with glucose dehydrogenase fromBacillus megaterium, wherein the regeneration of cofactor NADPH occurswith the aid of the glucose dehydrogenase and with glucose as acosubstrate.

It is the object of the invention to provide a process which enables theeconomical production of enantiomerically pure diols of general formulaII in high yields and with high enantiomeric purity without anybyproducts.

According to the invention, this object is achieved by a process of theinitially mentioned kind, wherein the oxidoreductase is reduced in thepresence of NADH or NADPH as a cofactor and which is characterized inthat

a) the hydroxy ketone is provided in the reaction at a concentration of≧50 g/l,

b) the oxidized cofactor NAD or NADP having formed is regeneratedcontinuously by oxidation of a secondary alcohol of general formulaR_(X)R_(Y)CHOH, wherein R_(X), R_(Y) independently represent hydrogen,branched or unbranched C₁-C₈-alkyl and C_(total)≧3, and

c) the reduction of the hydroxy ketone and the oxidation of thesecondary alcohol are catalyzed by the same oxidoreductase.

A preferred embodiment of the process is characterized in that theoxidoreductase

a) comprises an amino acid sequence in which at least 50% of the aminoacids are identical to those of amino acid sequence SEQ ID NO:1, SEQ IDNO:8, SEQ ID NO: 11 or SEQ ID NO: 14,

b) is encoded by the nucleic acid sequence SEQ ID NO:2, SEQ ID NO:3, SEQID NO:9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 15,or

c) is encoded by a nucleic acid sequence which hybridizes to SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:13 or SEQ ID NO: 15 under stringent conditions.

According to the invention, the above-mentioned object is also achievedby a process of the initially mentioned kind, wherein the oxidoreductase

a) comprises an amino acid sequence in which at least 50% of the aminoacids are identical to those of amino acid sequence SEQ ID NO: 1, SEQ IDNO:8, SEQ ID NO: 11 or SEQ ID NO:14,

b) is encoded by the nucleic acid sequence SEQ ID NO:2, SEQ ID NO:3, SEQID NO:9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 15,or

c) is encoded by a nucleic acid sequence which hybridizes to SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13or SEQ ID NO:15 under stringent conditions.

It has been found that the polypeptides comprising amino acid sequencesSEQ ID NO: 1, SEQ ID NO:8 and SEQ ID NO: 11 show oxidoreductase activityand can be used for reducing tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate with a diastereomeric excessof >99% to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. Otherhydroxy ketones of general formula I can be reduced in the same mannerby means of amino acid sequences SEQ ID NO:1, SEQ ID NO:8 and SEQ IDNO:11.

In addition, the aforementioned oxidoreductases have the advantage ofbeing able to regenerate the oxidized cofactor that is formed during thereduction by reducing a secondary alcohol. Thus, a particular economicadvantage of said oxidoreductases also consists in that, in contrast toprocesses of the prior art (U.S. Pat. No. 6,645,746 B and EP 0 569 998B), no further enzyme is required for cofactor regeneration.

A DNA sequence SEQ ID NO:2, which codes for a polypeptide comprising SEQID NO:1, is obtainable, for example, from the genome of the organismRubrobacter xylanophilus DSM 9941. In addition, it has been found thatthe DNA sequence SEQ ID NO:3 can be used for expressing the polypeptideof SEQ ID NO:1 in Escherichia.

A DNA sequence SEQ ID NO:9, which codes for a polypeptide comprising SEQID NO:8, is obtainable, for example, from the genome of the organismGeobacillus thermodenitrificans DSM 465. In addition, it has been foundthat the DNA sequence SEQ ID NO:10 can be used for expressing thepolypeptide of SEQ ID NO:8 in Escherichia.

A DNA sequence SEQ ID NO:12, which codes for a polypeptide comprisingSEQ ID NO:11, is obtainable, for example, from the genome of theorganism Chloroflexus aurantiacus DSM 635.

A DNA sequence SEQ ID NO:15, which codes for a polypeptide comprisingSEQ ID NO:14, is obtainable, for example, from the organism Candidamagnoliae CBS 6396.

Thus, the present invention also relates to a process for the reductionof hydroxy ketones of general formula I into diols of general formulaII, using a polypeptide comprising amino acid sequence SEQ ID NO:1, SEQID NO:8, SEQ ID NO:11 or SEQ ID NO:14, or a polypeptide which comprisesan amino acid sequence which is identical by at least 50% to the aminoacid sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:1 or SEQ ID NO:14,i.e., a polypeptide which can be derived by substitution, insertion,deletion or addition from at least one amino acid of SEQ ID NO:1, SEQ IDNO: 8, SEQ ID NO:11 or SEQ ID NO:14, or using a polypeptide which isencoded by the nucleic acid sequence SEQ ID NO:2, SEQ ID NO:9, SEQ IDNO:12 or SEQ ID NO:15 or by a nucleic acid sequence which hybridizes toSEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:12 or SEQ ID NO:15 under stringentconditions.

By a nucleic acid sequence which hybridizes, for example, to SEQ ID NO:2under stringent conditions, a polynucleotide is understood which can beidentified via the colony hybridization method, the plaque hybridizationmethod, the Southern hybridization method or comparable methods, usingSEQ ID NO:2 as a DNA probe.

For this purpose, the polynucleotide immobilized on a filter ishybridized, for example, to SEQ ID NO:2 in a 0.7-1 M NaCl solution at60° C. Hybridization is carried out as described, for instance, inMolecular Cloning, A Laboratory Manual, Second Edition (Cold SpringHarbor Laboratory Press, 1989) or in similar publications. Subsequently,the filter is washed with a 0.1 to 2-fold SSC solution at 65° C.,wherein a 1-fold SSC solution is understood to be a mixture consistingof 150 mM NaCl and 15 mM sodium citrate.

In the process according to the invention, the polypeptide comprisingthe sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14 andpolypeptides derivable from said polypeptides, respectively, can be usedeither in a completely purified state, in a partially purified state oras cells containing the polypeptide SEQ ID NO:1, SEQ ID NO:8, SEQ IDNO:11 or SEQ ID NO:14. Thereby, the cells used can be provided in anative, permeabilized or lysed state. Preferably, the polypeptidecomprising the sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ IDNO:14 and derivatives derivable therefrom, respectively, areoverexpressed in a suitable host organism such as, for example,Escherichia coli, and the recombinant polypeptide is used for reducingthe hydroxy ketone of general formula I.

5.000 to 10 Mio U of oxidoreductase SEQ ID NO:1, SEQ ID NO:8, SEQ IDNO:11 or SEQ ID NO:14 or derivatives thereof, respectively, are used perkg of the compound of Formula I to be reacted (upwardly open). Herein,the enzyme unit 1 U corresponds to the enzyme amount which is requiredfor reacting 1 μmol of the hydroxy ketone of formula I per minute (min).

The enzymatic reduction according to the invention proceeds under mildconditions so that the degradation of the frequently unstable hydroxyketone and thus the formation of undesired byproducts can be largelyavoided. The process according to the invention has a high service lifeand a diastereomeric purity of normally >99% of the chiral diolsproduced.

A preferred embodiment of the invention is characterized in that thecofactor used in the process is continuously reduced with a cosubstrate.Preferably, NAD(P)H is used as the cofactor, with the NAD(P) formed inthe reduction again being reduced to NAD(P)H by means of thecosubstrate.

Secondary alcohols such as 2-propanol, 2-butanol, 2-pentanol,3-pentanol, 4-methyl-2-pentanol, 2-heptanol, 2-octanol or cyclohexanolare thereby preferably used as cosubstrates. According to a particularlypreferred embodiment, 2-propanol is used for coenzyme regeneration. Theamount of cosubstrate for the regeneration can range from 5 to 95% byvolume, based on the total volume.

Coenzyme regeneration is, for example, likewise effected via thepolypeptide comprising SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ IDNO:14.

In the process according to the invention, the hydroxy ketone of generalformula I is preferably used in an amount of from 5 to 50% by weight (50g/l to 50 g/l), based on the total volume, preferably from 8 to 40% byweight, in particular from 10 to 25% by weight.

A particularly preferred embodiment is characterized in that tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate, tert. butyl(5S)-6-chloro-5-hydroxy-3-oxohexanoate or tert. butyl(5S)-5,6-dihydroxy-3-oxohexanoate is used as the hydroxy ketone ofgeneral formula (I).

Preferably, the process according to the invention is carried out in anaqueous organic two-phase system.

The aqueous portion of the reaction mixture in which the enzymaticreduction proceeds preferably contains a buffer, e.g., a potassiumphosphate, tris/HCl or triethanolamine buffer, having a pH value of from5 to 10, preferably a pH of from 6 to 9. In addition, the buffer cancontain ions for stabilizing or activating the enzymes such as, forexample, zinc ions or magnesium ions.

While carrying out the process according to the invention, thetemperature suitably ranges from about 10° C. to 70° C., preferably from20° C. to 45° C.

In a further preferred embodiment of the process according to theinvention, the enzymatic conversion is carried out in the presence of anorganic solvent which is not miscible with water or is miscible withwater only to a limited degree. Said solvent is, for example, asymmetric or unsymmetric di(C₁-C₆)alkyl ether, a linear-chain orbranched alkane or cycloalkane or a water-insoluble secondary alcoholwhich, at the same time, represents the cosubstrate. The preferredorganic solvents are diethyl ether, tertiary butyl methyl ether,diisopropyl ether, dibutyl ether, butyl acetate, heptane, hexane,2-octanol, 2-heptanol, 4-methyl-2-pentanol and cyclohexanol. In case ofthe last-mentioned secondary alcohols, the solvent can simultaneouslyalso serve as a cosubstrate for cofactor regeneration.

If water-insoluble solvents and cosubstrates, respectively, are used,the reaction batch consists of an aqueous phase and an organic phase.According to its solubility, the hydroxy ketone is distributed betweenthe organic phase and the aqueous phase. In general, the organic phasehas a proportion of from 5 to 95%, preferably from 10 to 90%, based onthe total reaction volume. The two liquid phases are preferably mixedmechanically so that, between them, a large surface area is generated.Also in this embodiment, the NAD formed in the enzymatic reduction, forexample, can again be reduced to NADH with a cosubstrate, such asdescribed above.

The concentration of the cofactor, in particular of NADH or NADPH,respectively, in the aqueous phase generally ranges from 0.001 mM to 10mM, in particular from 0.01 mM to 1 mM.

In the process according to the invention, a stabilizer ofoxidoreductase/dehydrogenase can also be used. Suitable stabilizers are,for example, glycerol, sorbitol, 1,4-DL-dithiothreitol (DTT) or dimethylsulfoxide (DMSO).

The process according to the invention is carried out, for example, in aclosed reaction vessel made of glass or metal. For this purpose, thecomponents are transferred individually into the reaction vessel andstirred under an atmosphere of, e.g., nitrogen or air.

According to another possible embodiment of the invention, the oxidizedcosubstrate (e.g. acetone) can be removed continuously and/or thecosubstrate (e.g. 2-propanol) can be newly added in a continuous mannerin order to shift the reaction equilibrium towards the reaction product(diol of general formula II).

In a further embodiment, the addition of the oxidoreductase according toSEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14 and/or of thecosubstrate can also be effected gradually in the course of the process.

After completion of the reduction, the reaction mixture is processed.For this purpose, the aqueous phase is optionally separated from theorganic phase and the organic phase containing the product is filtered.Optionally, the aqueous phase can also be extracted and processedfurther like the organic phase. Thereupon, the solvent is evaporatedfrom the organic phase and the diol of general formula II is obtained asa crude product. The crude product can then be purified further or useddirectly for the synthesis of a follow-up product. In the following, theinvention is illustrated further by way of examples.

EXAMPLE 1 Cloning of Oxidoreductase from Rubrobacter xylanophilus DSM9941

A) Cultivation of Rubrobacter xylanophilus DSM 9941

Cells of Rubrobacter xylanophilus DSM 9941 were cultivated in thefollowing medium at 50° C. (pH 7.2) and 140 rpm in a bacterial shaker:0.1% yeast extract, 0.1% tryptone, 0.004% CaSO₄×2H₂O, 0.02% MgCl₂×6H₂O,0.01% nitrilotriacetic acid, 100 ml phosphate buffer [5.44 g/l KH₂PO₄,43 g/l Na₂HPO₄×12 H₂O], 500 μl/10.01 M Fe citrate, 500 μl trace element[500 μl/l H₂SO₄, 2.28 g/l MnSO₄×H₂O, 500 mg/l ZnSO₄×7H₂O, 500 mg H₃BO₃,25 mg/l CuSO₄×5H₂O, 25 mg/l Na₂MoO₄×2H₂O, 45 mg/l CoCl₂×6H₂O]. On day 6of the cultivation, cells were separated from the culture medium bycentrifugation and stored at −80° C.

B) Amplification of the Gene Coding for Selective Oxidoreductase

Genomic DNA was extracted according to the method described in“Molecular Cloning” by Manniatis & Sambrook. The resulting nucleic acidserved as a template for the polymerase chain reaction (PCR) involvingspecific primers which were derived from the gene sequence publishedunder number 46106817 in the NCBI database. In doing so, the primerswere provided in a 5′-terminal position with restriction sites for theendonucleases Nde I and Hind III or Sph I, respectively (SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:7), for subsequent cloning into an expressionvector.

Amplification was carried out in a PCR buffer [10 mM tris-HCl, (pH 8.0);50 mM KCl; 10 mM MgSO₄; 1 mM dNTP Mix; per 20 pMol of primer and 2.5 Uof Platinum Pfx DNA polymerase (Invitrogen)] with 500 ng of genomic DNAand the following temperature cycles:

Cycle 1: 94° C., 2 min

Cycle 2×30: 94° C., 15 sec

-   -   54° C., 30 sec    -   68° C., 60 sec        Cycle 3: 68° C., 7 min    -   4° C., ∞

The resulting PCR product having a size of about 750 bp was restrictedafter purification over a 1% agarose gel with the aid of theendonucleases Nde I and Hind III or Sph I and Hind III, respectively,and was ligated into the backbone of the pET21a vector (Novagen) or ofthe pQE70 vector (Qiagen), respectively, which backbone had been treatedwith the same endonucleases. After transforming 2 μl of the ligationbatch into E. coli Top 10 F′ cells (Invitrogen), plasmid DNA ofampicillin-resistant colonies was tested for the presence of an inserthaving a size of 750 bp by means of a restriction analysis with theendonucleases Nde I and Hind III or Sph I and Hind III, respectively.Plasmid preparations from the clones which were positive for thefragment were subjected to a sequence analysis and subsequentlytransformed into Escherichia coli BL21 Star (Invitrogen) and E. coliRB791 (genetic stock, Yale), respectively.

EXAMPLE 2 Efficient Expression of Polypeptide SEQ ID NO:1 in Escherichiacoli Cells

In order to obtain efficient expression of the polypeptide SEQ ID NO:1in Escherichia coli cells, for cloning into an expression vector codingDNA SEQ ID NO:3 was used as a template in a PCR reaction. In the regionof the first 160 base pairs, this DNA sequence differed in 51 bases fromthe previously known DNA sequence (SEQ ID NO:2). This modification wasconservative and did not result in a change in the amino acid sequence.

Amplification was carried out in a PCR buffer [10 mM tris-HCl, (pH 8.0);50 mM KCl; 10 mM MgSO₄; 1 mM dNTP Mix; per 20 pMol of primer (SEQ IDNO:6, SEQ ID NO:5) and 2.5 U of Platinum Pfx DNA polymerase(Invitrogen)] with 50 ng of DNA SEQ ID NO:3 as a template and thefollowing temperature cycles:

Cycle 1: 94° C., 2 min

Cycle 2×30: 94° C., 40 sec

-   -   56° C., 30 sec    -   68° C., 60 sec        Cycle 3: 68° C., 7 min    -   4° C., ∞

The resulting PCR product having a size of about 750 bp was ligatedafter purification over a 1% agarose gel with the aid of theendonucleases Nhe I and Hind III into the backbone of the pET21a vector(Novagen), which backbone had been treated with the same endonucleases.

After transforming 2 μl of the ligation batch into E. coli Top 10 F′cells (Invitrogen), plasmid DNA of ampicillin-resistant colonies wastested for the presence of an insert having a size of 750 bp by means ofa restriction analysis with the endonucleases Nhe I and Hind III.Plasmid preparations from the clones which were positive for thefragment were subjected to a sequence analysis and subsequentlytransformed into Escherichia coli BL21 Star (Invitrogen).

EXAMPLE 3 Preparation of Oxidoreductase from Rubrobacter xylanophilusDSM 9941

The Escherichia coli strains BL21 Star (Invitrogen, Karlsruhe, Germany)and RB791 (E. coli genetic stock, Yale, USA), respectively, which hadbeen transformed with the expression construct, were cultivated in amedium (1% tryptone, 0.5% yeast extract, 1% NaCl) with ampicillin (50μg/ml) until an optical density of 0.5, measured at 550 nm, was reached.The expression of recombinant protein was induced by addingisopropylthiogalactoside (IPTG) at a concentration of 0.1 mM. 16 hoursafter the induction at 25° C. and 220 rpm, the cells were harvested andfrozen at −20° C.

For enzyme recovery, 30 g cells were suspended in 150 ml oftriethanolamine buffer (100 mM, pH=7.2 mM MgCl₂, 10% glycerol) andbroken down by means of a high-pressure homogenizer. Subsequently, theenzyme solution was mixed with 150 ml of glycerol and stored at −20° C.

The enzyme solution thus obtained was used for the synthesis of tert.butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate.

In the diluted state, the enzyme solution obtained was also used for thecorresponding enzymatic measurements. Thereby, the activity test wasmade up of the following: 870 μl of 100 mM TEA buffer, pH 7.0, 160 μgNAD(P)H, 10 μl diluted cell lysate. The reaction was initiated by adding100 μl of the 100 mM substrate solution tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate to the reaction mixture.

EXAMPLE 4 Conversion of tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate by means of oxidoreductase SEQ IDNO:1

For the conversion of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoateinto tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of900 μl buffer (100 mM TEA, pH=7.1 mM MgCl₂), 100 μl 2-propanol, 10 μltert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product(enantiomeric purity >99%), 0.1 mg NAD and 100 μl enzyme suspension (seeExample 3) was incubated in an Eppendorf reaction vessel for 24 h atroom temperature, under continual mixing. After 24 h, 96% of the tert.butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had been reduced totert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. The diastereomericexcess amounted to >99%.

The determination of the conversion as well as of the diastereomericexcess was performed by chiral gas chromatography. For this purpose, agas chromatograph GC-17A from Shimadzu comprising a chiral separatingcolumn CP-Chirasil-DEX CB (Varian Chrompack, Darmstadt, Germany), aflame ionization detector and helium as a carrier gas was used.

The separation of tert. butyl-6-cyano-3,5-dihydroxyhexanoate occurred at0.72 bar and for 10 min at 50° C., 5° C./min→200° C. for 10 min.

The retention times were: (R-BCH) 4.4 min; (R,R-BCH) 47.1 min and(R,S-BCH) 48.2 min.

EXAMPLE 5 Synthesis of tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate from tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate by means of oxidoreductase SEQ IDNO:1

For a further conversion of tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of 550 μl buffer (100mM TEA, pH=7.1 mM MgCl₂), 150 μl 2-propanol, 200 μl tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product (enantiomericpurity >99%), 0.1 mg NAD and 200 μl enzyme suspension (see Example 3)was incubated in an Eppendorf reaction vessel. In the course of thereaction, the acetone/2-propanol mixture formed was evaporatedrepeatedly by the introduction of nitrogen, and fresh 2-propanol and 50μl of enzyme were added. After 2 to 3 repeats at 24-hour intervals, >90%of the tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had beenreduced to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. Thediastereomeric excess amounted to >99%.

EXAMPLE 6 Synthesis of tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate from tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate by means of oxidoreductase SEQ IDNO:1

For a further conversion of tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of 6.7 ml buffer (100mM TEA, pH=9), 1.7 ml 2-propanol, 2 ml tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product (enantiomericpurity >99%), 1.0 mg NAD and 150 mg frozen cells E. coli BL21 Star,containing oxidoreductase SEQ ID NO:1, (see Example 3) was incubated ina reaction vessel at 45° C. After 24 h, >90% of the tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate used had been reduced to tert.butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. The diastereomeric excessamounted to >99%.

EXAMPLE 7 Synthesis of tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate from tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate by means of oxidoreductase SEQ IDNO:8

For a further conversion of tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of 5.0 ml buffer (100mM TEA, pH=7.5), 2.0 ml 2-propanol, 4.0 ml tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product (enantiomericpurity >99%), 1.0 mg NAD and 250 mg frozen cells E. coli RB 791,containing oxidoreductase SEQ ID NO:8, (corresponding to Examples 2 and3) was incubated in a reaction vessel at 40° C. After 24 h, >90% of thetert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had been reducedto tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. Thediastereomeric excess amounted to >99%.

EXAMPLE 8 Synthesis of tert. butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate from tert. butyl(5R)-6-cyano-5-hydroxy-3-oxohexanoate by means of oxidoreductase SEQ IDNO:11

For the conversion of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoateinto tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of350 μl buffer (100 mM potassium phosphate, pH=7), 150 μl 2-propanol, 50μl tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product(enantiomeric purity >99%), 0.025 mg NAD and 15 μl enzyme suspension SEQID NO:11 (see Example 3) was incubated in an Eppendorf reaction vesselfor 48 h at room temperature, under continual mixing. After 48 h, >80%of the tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had beenreduced to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. Thediastereomeric excess amounted to >99%.

The invention claimed is:
 1. A process for the enantioselectiveenzymatic reduction of a hydroxy ketone of general formula I

wherein R₁=C₁-C₆ alkyl and R₂=—Cl, —CN, —OH, —H or C₁-C₆ alkyl, to achiral diol of general formula II

wherein R₁ and R₂ have the same meaning as in formula I, wherein thehydroxy ketone is reduced with an oxidoreductase in the presence of acofactor, wherein the oxidoreductase a) comprises the amino acidsequence of SEQ ID NO:1, b) is encoded by the nucleic acid sequence SEQID NO:2 or SEQ ID NO:3, or c) is encoded by a nucleic acid sequencewhich hybridizes to SEQ ID NO:2 or SEQ ID NO:3 under stringentconditions comprising washing with 0.1-2.0×SSC solution at 65° C.
 2. Theprocess according to claim 1, wherein the cofactor is continuouslyreduced with a co-substrate.
 3. The process according to claim 1,wherein NAD(P)H is used as a cofactor.
 4. The process according to claim2, wherein at least one member selected from the group consisting of2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-heptanol and2-octanol is used as the co-substrate.
 5. The process according to claim1, wherein the hydroxy ketone is used in an amount of from 5 to 50% byweight based on the total reaction volume.
 6. The process according toclaim 1, wherein at least one member selected from the group consistingof tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate, tert. butyl(5S)-6-chloro-5-hydroxy-3-oxohexanoate and tert. butyl(5S)-5,6-dihydroxy-3-oxohexanoate is used as the hydroxy ketone ofgeneral formula (I).
 7. The process according to claim 1, wherein theTTN (total turn over number=mol of reduced hydroxy ketone/mol ofcofactor used) is ≦10³.
 8. The process according to claim 1, wherein theprocess is carried out in an aqueous organic two-phase system.
 9. Theprocess according to claim 1, wherein, in addition, at least one organicsolvent selected from the group consisting of diethyl ether, tertiarybutyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate,butyl acetate, heptane, hexane and cyclohexane is used.
 10. A processfor the enantioselective enzymatic reduction of a hydroxy ketone ofgeneral formula I

wherein R₁=C₁-C₆ alkyl and R₂=—Cl, —CN, —OH, —H or C₁-C₆ alkyl, to achiral diol of general formula II

wherein R₁ and R₂ have the same meaning as in formula I, wherein thehydroxy ketone is reduced with an oxidoreductase in the presence of acofactor, wherein the oxidoreductase comprises the amino acid sequenceSEQ ID NO:1.
 11. The process according to claim 1, wherein the hydroxyketone is used in an amount of from 8-40% by weight, based on the totalreaction volume.
 12. The process according to claim 1, wherein thehydroxy ketone is used in an amount of from 10-25% by weight, based onthe total reaction volume.
 13. A process for the enantioselectiveenzymatic reduction of a hydroxy ketone of general formula I

wherein R₁=C₁-C₆ alkyl and R₂=—Cl, —CN, —OH, —H or C₁-C₆ alkyl, to achiral diol of general formula II

wherein R₁ and R₂ have the same meaning as in formula I, wherein thehydroxy ketone is reduced with an oxidoreductase in the presence of acofactor, wherein said oxidoreducase is encoded by a nucleic acidsequence which hybridizes to SEQ ID NO:2 or SEQ ID NO:3 under stringentconditions comprising washing with 0.1-2.0×SSC solution at 65° C.